Folding Mechanisms of Dihydrofolate Reductase and the Response Regulators

二氢叶酸还原酶的折叠机制及其响应调节剂

• 批准号: 0721312
• 负责人: C Robert Matthews
• 金额: $ 57万
• 依托单位: University of Massachusetts Medical School
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0721312&HistoricalAwards=false
• 关键词: Folding; Mechanisms; Dihydrofolate; Reductase; Response

The goal of this project is to perform a comparative analysis of the folding mechanisms of two members of the alpha/beta/alpha sandwich motif, one of the most common in biology. Previous NSF-supported studies of the mechanism of folding of one sub-class of this motif, dihydrofolate reductase (DHFR), have shown that this small two-domain protein folds via a sequential set of on-pathway partially-folded states in four parallel channels. By contrast, two members of the flavodoxin fold family, another sub-class of this motif, initially misfold to an off-pathway intermediate that must at least partially unfold to access the productive transition state leading to the native conformation. The molecular basis for this misfolding reaction will be probed by a combination of experimental and computational methods that will focus on determining the structural features and energetics of these intermediates for CheY, NtrC and Spo0F, all members of the response regulator sub-class of the flavodoxin fold. Continuous-flow (CF) small-angle x-ray scattering (SAXS), time-resolved Forster resonance energy transfer (trFRET) and far-UV circular dichroism (CD) measurements of microsecond folding reactions will provide quantitative information on dimensional properties and global secondary structure. Pulse-quench hydrogen exchange methodology and mass spectrometric analysis of mass-labeled peptides will enhance the circular dichroism data by identifying the segments that define the cores of stability. Mutational analysis will explore the roles of individual side chains in the folding and misfolding of CheY, with a particular focus on large nonpolar side chains in the pair of hydrophobic cores on either side of the central beta-sheet. The role of chain entropy in the CheY misfolding reaction will be tested by creating permutations that vary the chain connectivity while preserving 3D structure. Collaborative native-centric simulations of response regulator folding mechanisms will provide detailed structural insights into the misfolded species and the process by which they backtrack to the native conformations. Comparisons with the results for the on-pathway sub-millisecond folding reaction in DHFR will provide additional insights into the contrast with response regulators. The results of this multi-dimensional comparative analysis are expected to enhance the understanding of early folding reactions in alpha/beta/alpha sandwich proteins and the basis for the misfolding reactions in the flavodoxin fold.The broader impact of this project has training, educational and technology development components. Undergraduate, graduate and postdoctoral fellows will receive training in molecular biophysics, including spectroscopic methods, thermodynamics and kinetics, sophisticated data analysis, protein engineering, and the protein folding problem. Several of the methods employed, including circular dichroism and fluorescence spectroscopy, and data analysis algorithms developed will form a part of a new advanced topics course in Biophysical Methods for graduate students in the PI's Department. The analytical tools developed during the course of this research on protein folding mechanisms are currently being used in over a dozen research labs around the world, and will be made more accessible via a website sponsored by the Biochemistry and Molecular Pharmacology Department. The continuous-flow mixing technology developed during the previous grant period will continue to be optimized and available to all users by collaboration (for CF-trFRET and CF-CD) or through the BioCAT beamline at the Advanced Photon Source at Argonne National Laboratory (for CF-SAXS).

该项目的目标是对生物学中最常见的α/β/α三明治基序的两个成员的折叠机制进行比较分析。 先前NSF支持的对该基序的一个亚类二氢叶酸还原酶（DHFR）的折叠机制的研究表明，这种小的双结构域蛋白质在四个平行通道中通过一组连续的通路上部分折叠状态折叠。 相比之下，黄素氧还蛋白折叠家族的两个成员，该基序的另一个亚类，最初错误折叠为必须至少部分展开以进入导致天然构象的生产性过渡态的非途径中间体。 这种错误折叠反应的分子基础将通过实验和计算方法的组合来探测，这些方法将集中于确定CheY，NtrC和Spo 0 F的这些中间体的结构特征和能量，这些中间体是flavodoxin折叠的响应调节子类的所有成员。 连续流（CF）小角度X射线散射（SAXS），时间分辨福斯特共振能量转移（trFRET）和远紫外圆二色性（CD）测量微秒折叠反应将提供定量信息的尺寸属性和全球二级结构。 脉冲淬灭氢交换方法和质谱分析的质量标记的肽将提高圆二色谱数据，通过确定的部分，定义的稳定性的核心。 突变分析将探索单个侧链在CheY折叠和错误折叠中的作用，特别关注中心β折叠两侧疏水核心中的大型非极性侧链。 Chey错误折叠反应中链熵的作用将通过创建改变链连接性同时保留3D结构的排列来测试。 合作的本地为中心的模拟反应调节器折叠机制将提供详细的结构的错误折叠的物种和他们回溯到本地构象的过程的见解。 与DHFR中通路上亚毫秒折叠反应的结果的比较将提供与响应调节剂的对比的额外见解。 这一多维比较分析的结果预计将加强对α/β/α夹心蛋白早期折叠反应的理解，以及对flavodoxin fold.The更广泛的影响，该项目的培训，教育和技术开发组件的错误折叠反应的基础。 本科生，研究生和博士后研究员将接受分子生物物理学的培训，包括光谱方法，热力学和动力学，复杂的数据分析，蛋白质工程和蛋白质折叠问题。 所采用的几种方法，包括圆二色谱和荧光光谱，以及开发的数据分析算法将成为PI系研究生生物物理方法新的高级主题课程的一部分。 在蛋白质折叠机制研究过程中开发的分析工具目前正在世界各地的十几个研究实验室中使用，并将通过生物化学和分子药理学系赞助的网站更容易访问。 在上一个资助期内开发的连续流混合技术将继续得到优化，并通过合作（CF-trFRET和CF-CD）或通过阿贡国家实验室高级光子源的BioCAT光束线（CF-SAXS）提供给所有用户。